Contact PD/PI: Sitaraman, Divya Summary Sleep and arousal regulate key behavioral and physiological processes. Inadequate sleep or impaired sleep-wake transitions lead to a range of cognitive, attentional, motor, and emotional deficits. Treatment and management of these conditions will benefit greatly from an understanding of how sleep-wake transitions are controlled and how the homeostatic sleep drive is encoded in the nervous system. A long term goal of this proposal is to understand the precise circuit mechanisms underlying the neural control of sleep, and how this circuit is regulated by behavioral drive. The goal of this proposal is to exploit Drosophila as an experimental system in which we can mechanistically dissect the neuronal underpinnings of the sleep control network and identify mechanisms by which conserved neuromodulators like dopamine regulate sleep. Technical challenges that have limited prior findings in this area are lack of cell type specific tools and inability to physiologically map circuits with accuracy. To surmount these challenges, we will combine the unique cell-specific neurogenetic manipulability of the Drosophila model system with an array of behavioral and novel neurophysiological approaches. In pursuit of identifying and characterizing dopamine regulation of sleep control network in Drosophila we have proposed three specific aims. In Aim 1, we will identify the functional connectivity between dopamine neurons and key sleep- and wake- promoting neurons within the associative neural network of mushroom body. By reciprocal activation and blocking of functionally upstream and downstream neurons we will map how the sleep and arousal information flow occurs within the identified sleep regulating neurons. In Aim 2, we will employ direct physiological mapping tools to identify functional connectivity within sleep regulating neurons by stimulating one neural node while recording from the other using genetically encoded calcium and voltage indicators. Finally, in Aim 3 we will test if activity of the sleep-regulating dopamine neurons is influenced by altered sleep-drive. Additionally, we will characterize the nature of synaptic inputs to the dopamine neurons using techniques refined and validated in Aims 1 and 2. The combination of novel genetic, behavioral and physiological approaches and successful completion of these Aims will address questions of great relevance to mammalian sleep circuit that have been plagued by technical challenges. These include the role of dopamine in modulating activity of sleep regulating neurons, synaptic plasticity mechanisms underlying the sleep-wake switch, and the cellular encoding of sleep drive. Project Summary/Abstract Page 6